Journal: Nature Communications

Article Title: Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish

doi: 10.1038/s41467-023-37026-6

Figure Lengend Snippet: a Strategy for the generation of Ara and Arb KI medaka strains. We designed a gRNA targeting ara intron 8 and arb intron 7 (the last intron). In the donor plasmids, we cloned a genomic fragment beginning from the end of exon 8 of ara and exon 7 of arb and ending just before the stop codon in the last exon, exon 9 of ara , and exon 8 (shown as black closed boxes with ‘E8’) of arb , where the 3xFLAG sequences (shown as blue boxes) and a P2A (2A peptide from porcine teschovirus-1)-mClover3 cassette (shown as green boxes) was placed in the frame. Thus, endogenous Ara and Arb were expressed as FLAG fusion proteins. Both AR-FLAG and P2A-mClover3 were expected to be expressed under the control of the endogenous Ar promoter. To generate KI medaka, sgRNA (for genome digestion in the final intron), donor plasmid, and Cas9 mRNA were co-injected into one-cell-stage medaka embryos. After injection, concurrent cleavage of the targeted genomic locus and the donor plasmid resulted in the integration of donor plasmid DNA containing 3xFLAG-T2A-mClover3 by non-homologous end joining (NHEJ). The scheme shows the forward integration of 3xFLAG-T2A-mClover3. b Expression of mClover3 in adult males of the two knock-in medaka strains, ara FLAG-2A-mClover3 (Ara-KI) and arb FLAG-2A-mClover3 (Arb-KI). White, grey, and red arrows indicate the regions adjacent to pectoral, dorsal, and anal fins, respectively (Ara-KI). White, grey, and red arrows indicate the pectoral, dorsal, and anal fins, respectively (Arb-KI). c Immunohistochemical detection of FLAG-tagged endogenous Ara and Arb in longitudinal sections of papillary processes of the anal fin (6 μm thickness). The merged images represent red fluorescence for immunostaining of FLAG (anti-DDDDK-tag mouse mAb monoclonal antibody), green fluorescence for immunostaining of mClover3 (anti-GFP D5.1XP rabbit mAb monoclonal antibody), and blue fluorescence for nuclear staining by DAPI. The medaka Arb-FLAG, but not Ara-FLAG, translocated into the nuclei of cells located in the distal tip of a bone nodule of papillary processes (marked by white arrows). d Representative micrographs of Masson/trichrome staining and immunohistochemical detection of FLAG-tagged endogenous Ara and Arb in adjacent sections of the urogenital region (8 μm thickness). Nuclear localisation of Ara-FLAG and Arb-FLAG was observed in the medulla ventral to the sperm duct, where ar DKO exhibited hyperplasia. e, f Representative micrographs showing immunohistochemical detection of FLAG-tagged endogenous Ara and Arb, and DAPI counterstaining in the brain (12 μm thickness). f shows a higher magnification of the POA. Nuclear localisation of both Ara-FLAG and Arb-FLAG was observed in the POA. n = 6 for Ara-KI and Arb-KI males, respectively.

Article Snippet: Briefly, after antigen retrieval by incubating slides in 0.1 mM citrate buffer (pH 6.0) in an autoclave (121 °C) for 1 min, the sections were blocked for 1 h using 1xPBS containing 0.1% Tween 20, 2% BSA, and 2% foetal bovine serum, and incubated with anti-DDDDK-tag (FLAG) mouse mAb monoclonal antibody (M185-3L, MBL, Nagoya, Japan, 1:300 dilution) and anti-GFP D5.1XP rabbit mAb monoclonal antibody having cross-reactivity to the mClover3 (#2956, Cell Signaling, Danvers, MA, USA, 1:300 dilution) overnight at 4 °C.

Techniques: Clone Assay, Plasmid Preparation, Injection, Non-Homologous End Joining, Expressing, Knock-In, Immunohistochemical staining, Fluorescence, Immunostaining, Staining

Journal: Life Science Alliance

Article Title: The ERK activator, BCI, inhibits ciliogenesis and causes defects in motor behavior, ciliary gating, and cytoskeletal rearrangement

doi: 10.26508/lsa.202301899

Figure Lengend Snippet: Tools and reagents.

Article Snippet: GFP (D5.1) rabbit mAb , Cell Signaling Technology , 2956S.

Techniques: Staining, Mutagenesis

Journal: Cell reports

Article Title: A multiplexed in vivo approach to identify driver genes in small cell lung cancer

doi: 10.1016/j.celrep.2023.111990

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-GFP (D5.1) Monoclonal , Cell Signaling Technology , Cat#2956; RRID:AB_1196615.

Techniques: Plasmid Preparation, Recombinant, Modification, Labeling, Amplification, Staining, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Western Blot, DNA Extraction, Purification, Sequencing, Generated, Software

Journal: The EMBO Journal

Article Title: AATF /Che‐1 localizes to paraspeckles and suppresses R‐loops accumulation and interferon activation in Multiple Myeloma

doi: 10.15252/embj.2021109711

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal anti‐GFP (D5.1) (WB 1:500) , Cell Signaling , Cat# 2956.

Techniques: Isolation, Recombinant, Expressing, Plasmid Preparation, Luciferase, Dot Blot, Sequencing, Software

Journal: STAR Protocols

Article Title: Filter trapping protocol to detect aggregated proteins in human cell lines

doi: 10.1016/j.xpro.2022.101571

Figure Lengend Snippet: Representative results for specific aggregated proteins by immunoprobing (A) Filter trap assay of the indicated proteins expressed in HeLa S3 cells. The tagged proteins were detected by an anti-FLAG antibody. (B) Standard Western blot showing the levels of FLAG-tagged FTH1 and FTH1∗ proteins. Whole cell lysates were subfractionated by centrifugation into the supernatant and the pellet. (C) Filter trap assay of the indicated proteins expressed in HeLa S3 cells. The tagged proteins were detected with an anti-GFP antibody. (D) Filter trap assay of ubiquitinated proteins accumulated in HeLa S3 cells treated with MG132. The ubiquitinated proteins were detected using an anti-ubiquitin antibody.

Article Snippet: Rabbit monoclonal anti-GFP (D5.1) antibody (1:1,000) , Cell Signaling Technology , Cat#2956, RRID: AB_1196615.

Techniques: TRAP Assay, Western Blot, Centrifugation

Journal: STAR Protocols

Article Title: Filter trapping protocol to detect aggregated proteins in human cell lines

doi: 10.1016/j.xpro.2022.101571

Figure Lengend Snippet: Limitation of the protocol for cell lysis and freeze–thaw cycles (A) Different cell lysis methods were compared for the filter trap assay. Left panel: Staining of the total proteins on filter membrane. The lysate of HeLa S3 cells obtained after splicing modulation by SSA (100 ng/mL) for 24 h was used. Proteins on the membrane were detected by performing Revert 700 staining. Right panel: Filter trap assay of the indicated proteins expressed in HeLa S3 cells. The tagged proteins were detected using an anti-GFP antibody. (B) The effect of repeated freeze–thaw cycles was tested. The experiments were conducted as described in A, but the lysate were subjected to freeze–thaw cycles before samples were loaded onto the membrane.

Article Snippet: Rabbit monoclonal anti-GFP (D5.1) antibody (1:1,000) , Cell Signaling Technology , Cat#2956, RRID: AB_1196615.

Techniques: Lysis, TRAP Assay, Staining

Journal: STAR Protocols

Article Title: Filter trapping protocol to detect aggregated proteins in human cell lines

doi: 10.1016/j.xpro.2022.101571

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-GFP (D5.1) antibody (1:1,000) , Cell Signaling Technology , Cat#2956, RRID: AB_1196615.

Techniques: Recombinant, Protease Inhibitor, Staining, Blocking Assay, Software, Imaging

Journal: Cell Reports

Article Title: Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached

doi: 10.1016/j.celrep.2022.110995

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti- GFP (D5.1) XP , Cell Signaling , RRID: Cat# 2956.

Techniques: Recombinant, Transfection, Staining, Imaging, RNA Extraction, Over Expression, Software